1. Field
This disclosure is concerned generally with methods of detecting and/or determining the concentration of various substances found in fluids, especially human body fluids. More specifically, the disclosure is concerned with a method of determining such substances via solid phase immunoassay techniques.
2. Prior Art
The expression immunoassay, as used herein, refers to a method of determining the presence or concentration of a subtance in a fluid which method is based on the use of antibodies specific to that substance. Since it is known that antibodies to a given substance are extremely specific to that substance, research efforts have been directed in recent years to use that specificity in determining the presence or concentration of substances which are present in very small quantities in fluids, especially human body fluids such as blood. Although there now exists a wide variety of immunoassay techniques, the more common assays require the use of a label for either the antibody or the antigenic substance or hapten being determined. The use of a label permits a relatively rapid detection or quantitation in conventional laboratories using conventional equipment. A variety of labels are known and used in immunoassays. For example, fluorogenic materials useful in a fluoroimmunoassay (FIA) are described in U.S. Pat. No. 3,940,475, to Gross. Enzyme markers can be coupled to antibodies or antigens to perform an enzyme immunoassay (EIA) as illustrated in U.S. Pat. No. 3,654,090, to Schuurs et al. Radioisotopes can be incorporated into an antibody or substance to perform a radioimmunoassay (RIA) as illustrated in U.S. Pat. No. 3,555,153 to Axen et al. As used herein, the expression labeled antibody or its equivalent, includes any of those known labels.
A typical immunoassay requires, at some point, an immunochemical complexation between an antigenic substance and its respective antibody. Commonly, one of the species in such a complexation is labeled, and, by competing with, complexing with, or displacing an unknown substance in such complexation, and then quantitating the label (e.g. fluorometrically, enzymatically, radiometrically, etc.), it is possible to determine the unknown by known means. Prior to such quantitation, however, it is generally necessary to separate the immunochemically complexed products from the surrounding incubation medium. Such separations can be facilitated by providing one of species involved in an immobilized, insoluble, form. For example, it is known that antigenic substances, haptens, or antibodies thereto can be attached to, or incorporated in, various water insloluble carrier materials without substantial loss of biological activity. See for example, U.S. Pat. Nos. 3,555,153 (organic carriers) and 3,652,761 (inorganic carriers). When either of the reactants in an immunoassay is used in such an immobilized form, there is present a solid phase which, when appropriate, can be readily separated (e.g. by centrifugation) for label quantitation. The use of composites comprising antibodies or antigens associated with or immobilized on essentially water insoluble carrier materials is commonly referred to as a solid phase immunoassay (SPIA). As used herein, the expression immobilized antibody composite or the equivalent includes all forms of antibodies which have been attached to insoluble materials.
A wide variety of solid phase immunoassays have been developed in response to analytical needs and limitations. A number of these assays have been described in detail in Radioimmunoassay Methods, Ed. K. E. Kerkham & W. M. Hunter, Churchill Livingston, Edinburgh & London (1971). See specifically the chapter by L. Wide, "Solid Phase Antigen-Antibody Systems", pp. 405-413. In the above reference, there is described a so-called "sandwich" technique for the assay of antigens. See System 3 at p. 408. That system is based on the use of solid phase coupled antibodies and antibodies which have been labeled with a radioisotope. The antigen being assayed is initially complexed with the immobilized antibody by simply incubating the immobilized antibody composite with a fluid sample containing the antigen. The incubation period is about 24 hours. After the incubation period, the solid phase product is washed and then reacted with labeled antibodies which complex with the antigen already complexed on the solid phase antibodies. The uptake of labeled antibody (which can be conveniently counted) is directly correlated to the amount of antigen in the test solution. As noted in the above publication, the system is based on the use (and existence) of an antigen which can be bound to at least two antibodies simultaneously. Such antigens are referred to herein as polyvalent antigens and a variety of such antigens are known (e.g. hepatitis associated antigen or HAA, throid stimulating hormone or TSH, carcinoembryonic antigen or CEA, etc.).
Another system described in the above publication is the "Radiometric" assay (see System 6, p. 411). That assay, referred to as an immunoradiometric assay, involves incubating labeled antibodies with serum to be tested to complex antigens in the serum. After an incubation period, a solid phase coupled antigen is added to bind the remaining "free" antibodies. The radioactivity in the liquid phase is directly correlated with the amount of antigen in the sample. For maximum efficiency in this system, the antigens should have only one site capable of complexing with an antibody. Such antigens can be and are referred to herein as monovalent.
The present disclosure is based on our discovery that the certain features of known sandwich immunoassays such as that disclosed in System 3 of the cited publication (or recently issued U.S. Pat. No. 3,867,517 to Ling) and our own observations result in a novel sandwich immunoassay, the methodology of that assay being the reverse of that presently employed. By reversing the steps of known solid phase sandwich assays, we have found that a higher degree of sensitivity is possible and additionally tedius and time consuming intermediate wash step is eliminated. Details of our method are described hereunder.